fig4: Altering the potency of TLR4 responses enhances osteogenic functions. Osteoblasts cultured in three-dimensional collagen gels were either unstimulated or stimulated for one week with either 1 000 ng⋅mL−1 P3C or 1–1 000 ng⋅mL−1 of EcLPS (a–j). Additionally, mutant LPS structures namely LPxF, LPxE and WSK were purified as described in materials and methods and were employed to stimulate osteoblasts at a concentration of 1 μg⋅mL−1 (e–j). Regulation of ALP was determined through histochemical (a, e) and biochemical (b, f) methods and regulation of in vitro mineralization/calcium deposition was analyzed by histochemical (c, g) and biochemical (d, h) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. Expressions of IL-1β (i) and SHIP1 (j) were quantitated through real-time RT-PCR by generating a standard curve and through normalization with β-actin levels. Expression of ALP (a, b) and mineralization (c, d) were downmodulated with EcLPS in a dose-dependent manner. Relative to EcLPS, mutant LPS structures (LPxF, LPxE and WSK) were potent inducers of ALP (e, f) and in vitro mineralization (g, h). Real-time PCR data suggests that mutant LPS structures (LPxF, LPxE and WSK) were weak inducers of both IL-1β (i) and SHIP1 (j) relative to EcLPS-induced IL-1β (i) and P3C-induced SHIP1 (j). The experiments were repeated twice and representative data is shown. *P≤0.001, t-test; **P≤0.05, t-test.

Mentions:
In this study, we aimed to determine whether low doses of EcLPS have any positive influence on osteogenic functions. The data suggests that EcLPS employed at 1 ng⋅mL−1 moderately induced ALP (Figure 4a and 4b) and mineralization (Figure 4c and 4d) and this was downmodulated in a dose-dependent manner. We subsequently determined how mutant LPS structures that are weak TLR4 agonists (and therefore, weak inducers of inflammatory response) regulate osteogenic mediators. Mutant EcLPS structures namely, LPxF, LPxE and WSK were used along with P3C (positive control) and canonical EcLPS (negative control). When compared with EcLPS, osteoblasts stimulated with LPxF, LPxE or WSK significantly induced the expression of ALP (Figure 4e and 4f) and upregulated in vitro mineralization (Figure 5g and 5h). However, the induction of ALP and mineral deposition was more potent when osteoblasts were stimulated with P3C (Figure 5e and 5h). Moreover, SHIP1 was not significantly upregulated when osteoblasts were stimulated with any of the mutant LPS (i.e., LPxF, LPxE or WSK) relative to stimulating with P3C (Figure 5j). Despite lower induction of SHIP1, IL-1β was also not strongly induced by these mutant LPS structures relative to stimulation with the canonical EcLPS (Figure 5i). These results suggest that, irrespective of the potency of LPS structures that target TLR4, SHIP1 expression is not under the control of TLR4 signaling. Therefore, upregulation of osteogenic mediators in response to LPxF, LPxE or WSK, could be facilitated due to a weak inflammatory response mediated by these mutant LPS structures.

fig4: Altering the potency of TLR4 responses enhances osteogenic functions. Osteoblasts cultured in three-dimensional collagen gels were either unstimulated or stimulated for one week with either 1 000 ng⋅mL−1 P3C or 1–1 000 ng⋅mL−1 of EcLPS (a–j). Additionally, mutant LPS structures namely LPxF, LPxE and WSK were purified as described in materials and methods and were employed to stimulate osteoblasts at a concentration of 1 μg⋅mL−1 (e–j). Regulation of ALP was determined through histochemical (a, e) and biochemical (b, f) methods and regulation of in vitro mineralization/calcium deposition was analyzed by histochemical (c, g) and biochemical (d, h) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. Expressions of IL-1β (i) and SHIP1 (j) were quantitated through real-time RT-PCR by generating a standard curve and through normalization with β-actin levels. Expression of ALP (a, b) and mineralization (c, d) were downmodulated with EcLPS in a dose-dependent manner. Relative to EcLPS, mutant LPS structures (LPxF, LPxE and WSK) were potent inducers of ALP (e, f) and in vitro mineralization (g, h). Real-time PCR data suggests that mutant LPS structures (LPxF, LPxE and WSK) were weak inducers of both IL-1β (i) and SHIP1 (j) relative to EcLPS-induced IL-1β (i) and P3C-induced SHIP1 (j). The experiments were repeated twice and representative data is shown. *P≤0.001, t-test; **P≤0.05, t-test.

Mentions:
In this study, we aimed to determine whether low doses of EcLPS have any positive influence on osteogenic functions. The data suggests that EcLPS employed at 1 ng⋅mL−1 moderately induced ALP (Figure 4a and 4b) and mineralization (Figure 4c and 4d) and this was downmodulated in a dose-dependent manner. We subsequently determined how mutant LPS structures that are weak TLR4 agonists (and therefore, weak inducers of inflammatory response) regulate osteogenic mediators. Mutant EcLPS structures namely, LPxF, LPxE and WSK were used along with P3C (positive control) and canonical EcLPS (negative control). When compared with EcLPS, osteoblasts stimulated with LPxF, LPxE or WSK significantly induced the expression of ALP (Figure 4e and 4f) and upregulated in vitro mineralization (Figure 5g and 5h). However, the induction of ALP and mineral deposition was more potent when osteoblasts were stimulated with P3C (Figure 5e and 5h). Moreover, SHIP1 was not significantly upregulated when osteoblasts were stimulated with any of the mutant LPS (i.e., LPxF, LPxE or WSK) relative to stimulating with P3C (Figure 5j). Despite lower induction of SHIP1, IL-1β was also not strongly induced by these mutant LPS structures relative to stimulation with the canonical EcLPS (Figure 5i). These results suggest that, irrespective of the potency of LPS structures that target TLR4, SHIP1 expression is not under the control of TLR4 signaling. Therefore, upregulation of osteogenic mediators in response to LPxF, LPxE or WSK, could be facilitated due to a weak inflammatory response mediated by these mutant LPS structures.